Scientific Opinion on the substantiation of a health claim related to barley soup “Orzotto” and protection of blood lipids from oxidative damage

Presented by:

Jaturong Amonchaisup

Sophia Amenyah

Rafat KarbakhshPriscille Giner

Introduction / Background • The Applicant: Italsur srl, Italy• Product Name: “Orzotto”• Health Claim :“Orzotto contributes to protection of blood lipids from oxidative damage”. Daily inclusion of a portion of frozen barley soup “Orzotto” in a balanced and varied diet reduces oxidative damage of blood lipids.

• Target group: The general population

• Amount & Duration of consumption: 250 g barley soup “Orzotto” daily for two weeks.

Characterization of food or food components

Orzotto

Food Constituents

Pearled Barley

Tri-colored Swiss Chard Tuscan cabbage Bi-colored Spinach Savoy Cabbage

Purple carrotPotatoRed/Yellow Celery

Parsley onion

75% Pearled Barley

2.5%

2.5%

2.5%

2.5%

2.5%2.5%

2.5%2.5% 2.5% 2.5%

Pearled Barley

Tuscan black cabbage

Potatoes

Blue savoy cabbage

Purple carrot

Bi-colored Spinach

Tri-colored Swiss Chard

Onion

Yellow Celery

Red Celery

Parsley

Composition of Orzotto250 g Orzotto

• Health Claim: Contributes to the protection of blood lipids from oxidative damage

• Need to know:1) Mechanism of oxidative damage of lipid2) Bioactive compounds and their

mechanisms in preventing lipid oxidation.

Oxidative damage of lipid

1) Radical Formation

Fenton Rxn

2) Blood Lipid Oxidation

(Oxidized Lipid)

Atherosclerotic plaque

Bioactive compounds

• Potential bioactive compound: Antioxidants and radical scavenger.

• Major: Phenolic coumpounds• Minor: Tocopherol, Carotenoid, Ascorbic

acid, Selenium But, present in marginal amount

Phenolic compounds as antioxidant and radical

scavenger

1) Radical scavenging activity - Give H atom to peroxide radical - Peroxide radical Hydro peroxide (non-radical) - Phenolics Phenolic radicals (non-reactive)

Phenolic compounds as antioxidant and radical scavenger

2) Metal Sequestrant

Phenolics chelate with metal so metal become unavailable to initiate lipid oxidation

Quantification of phenolic compounds in

the food product

Total Phenolic compounds CompoundsIngredient

Proportion in Orzotto

(%)

Amount in Orzotto

(g/ 250 g)Major type of

phenolic compounds

Average Total Phenolic

compounds(mg / 100 g FW)

Amount Phenolic compounds in each

ingredient

Total Phenolic compounds in

food(mg/250 g)

Pearled Barley 75% 187.5 Phenolic Acids,

Proanthocyanidins 763.00 1,430.63

1,569

Tuscan black cabbage 2.5% 6.25 Quercetin, Phenolic

Acid 831.00 51.94

Potatoes 2.5% 6.25 Chlorogenic acid,

anthocyanins 33.33 2.08

Blue savoy cabbage 2.5% 6.25 Flavonoids 50.97 3.19

Purple carrot 2.5% 6.25

Anthocyanin, Hydroxycinnamic acid, syringic acids

74.64 4.67

Bi-colored Spinach 2.5% 6.25 Flavonoid: Quertecin,

Anthocyanin 278.35 17.40

Tri-colored Swiss Chard 2.5% 6.25

Flavonoid: Catechins, Kaempferol, Quertecin

192.00 12.00

Onion

2.5% 6.25Flavonoid: Quercetin, Isorhamnetin, Myricetin and Kaempferol

645.00 40.31

Celery 5% 12.5 Furanocoumarins,

bergapten, Apigenin 4.49 0.56

Parsley 2.5% 6.25 Flavonoids, Apigenin 103.70 6.48

Effect of Processing on Total Phenolic Content

Total phenolic compounds in 250 g Orzotto

(mg)

Loss during Process(Freezing + Thermal

process)

Remaining amount of phenolic compounds in 250 g Orzotto

(mg)

1,569 20 – 30 % 1,100

• Freezing– Causes minimal destruction – Sometimes, increases in the phenolic content

• Heating prior consumption– Few minutes minimal destruction – Sometimes, increases in the phenolic content

• Leaching– Major cause of loss– But, leached nutrients are still present in soup matrix

Other Bioactive compounds

Fenton Rxn

Vitamin C, Vitamin E, and Se as radical scavenger

Vitamin C & E

Se

Quantification of minor Bioactive compounds

Total Ascorbic AcidIngredient

Proportion in Orzotto

(%)

Amount in Orzotto

(g/ 250 g)

Average Ascorbic acid after cooking process

(mg / 100 g FW)

Amount of Ascorbic acid in each ingredient

Total Ascorbic acid

in food(mg/250 g)

Pearled Barley 75% 187.5 0 0.00

16.79

Tuscan black cabbage 2.5% 6.25 41 2.56

Potatoes 2.5% 6.25 13 0.81Blue savoy cabbage 2.5% 6.25 17 1.06

Purple carrot 2.5% 6.25 3.6 0.23Bi-colored Spinach 2.5% 6.25 9.8 0.61

Tri-colored Swiss Chard 2.5% 6.25 18 1.13

Onion 2.5% 6.25 5.2 0.33Celery 5% 12.5 6.1 0.76Parsley 2.5% 6.25 149 9.31

Total TocopherolIngredient

Proportion in Orzotto

(%)

Amount in Orzotto

(g/ 250 g)

Average Ascorbic acid after cooking process

(mg / 100 g FW)

Amount of Ascorbic acid in each ingredient

Total Ascorbic acid

in food(mg/250 g)

Pearled Barley 75% 187.5 0.01 0.02

1.19

Tuscan black cabbage 2.5% 6.25 0.85 0.05

Potatoes 2.5% 6.25 0.01 0.00Blue savoy cabbage 2.5% 6.25 0 0.00

Purple carrot 2.5% 6.25 1.03 0.06Bi-colored Spinach 2.5% 6.25 2.08 0.13

Tri-colored Swiss Chard 2.5% 6.25 1.89 0.12

Onion 2.5% 6.25 0 0.00Celery 5% 12.5 6.1 0.76Parsley 2.5% 6.25 0.7 0.04

Total SeleniumIngredient

Proportion in Orzotto

(%)

Amount in Orzotto

(g/ 250 g)

Average Ascorbic acid after cooking process

(µg / 100 g FW)

Amount of Ascorbic acid in each ingredient

Total Ascorbic acid

in food(µg/250 g)

Pearled Barley 75% 187.5 8.6 16.13

18.9

Tuscan black cabbage 2.5% 6.25 0.9 0.06

Potatoes 2.5% 6.25 0.3 0.02Blue savoy cabbage 2.5% 6.25 0.7 0.04

Purple carrot 2.5% 6.25 0.7 0.04Bi-colored Spinach 2.5% 6.25 6 0.38

Tri-colored Swiss Chard 2.5% 6.25 0.9 0.06

Onion 2.5% 6.25 0.6 0.04Celery 5% 12.5 1.0 0.13Parsley 2.5% 6.25 32.3 2.02

Total Amount of Bioactive Compound

250 g Orzotto

Bioactive Compound Amount

Phenolic compounds 1,100 mg

Ascorbic acid 16.79 mg

Tocopherol 1.19 mg

Selenium 18.9 µg

Scientific substantiation of the claimed effect

Human Clinical Studies:- Study proposed by applicant is unpublished Need a literature search on the effect of phenolic compounds in protecting blood lipid against oxidative damage.

- Literature search on PubMed, Cochrane Library, Web of knowledge and Google scholar

- Several RCT and clinical trials with significantly positive results were found.

Biomarkers used in the assessment of lipid oxidation

Direct measurement (based on EFSA documents)

• Decreased plasma oxidized LDL

• Decreased F2-isoprostanes (product from the peroxidation of essential fatty acid primarily arachidonic acid )

Indirect measurement (based on EFSA documents)

• Increased lag time before initiation of LDL oxidation

• Decreased plasma TBARS (Thiobarbituric acid reactive substances = by product of lipid peroxidation)

• Decreased plasma malondialdehyde (product of lipid peroxidation)

Summary of human clinical studies on the prevention of lipid oxidation by phenolic compounds

Phenolic compounds (>500 mg/day)Food Intake Quantity Duration Subjects Results

Pomegranate juice

570 – 1000 mg/day total

phenolic compounds

(mainly Flavonoids)

Average:2 weeks

Average: 13 healthy

subjects

Significant decrease in• Plasma oxidized LDL• F2-isoprostanes• Plasma TBARS• Lipid peroxide

Significant increase in• lag time before the initiation of LDL oxidation

Sweet potato leaves

Perilla

Tart cherry juice

Phenolic compounds (400-500mg/day)Food Intake Quantity Duration Subjects Results

Black tea

Avg. 460 mg/day total phenolic compounds

(mainly Flavonoids esp.

catechin)

2 – 8 weeks Average: 30 healthy

adults

Significant decrease in• Plasma oxidized LDL• Plasma MDA• Lipid peroxide

Significant increase in• lag time before the initiation of LDL oxidation

cranberry juice

Green tea polyphenol

extract

Catechin

Phenolic compounds (<400 mg/day)Food Intake Quantity Duration Subjects Results

Soy germ pasta

< 400 mg/day total phenolic compounds

(mainly Flavonoids)

Average:8 weeks

Average: 20 healthy

subjects

Significant decrease in• Plasma oxidized LDL• F2-isoprostanes• Lipid peroxide

Pycnogenol (Extract)

Cocoa powder

Grape-seedextract

According to these studies,

• Significant results in those clinical studies + Consistency of the results with similar studies carried out on the same food.

• The type of phenolic compounds that are available in our product are similar to those from studies (flavonoids such as quercetin, Proanthocyanidins and anthocyanin)

• And also they are all plant based product, so we expect to see more or less the same bioavailability.

• Our product provides 1,100 mg phenolic compound in each time intake, which is higher than those present in the most studies. So, we expect a good effect after 2 week of daily consumption.

• Also our product also provide 16.79 mg Vitamin C, 1.19 Vitamin E and 18.9 µg Selenium, which would have helping role in addition to the phenolic compounds.

Supporting Evidence

Literatures which show that each vegetable ingredient can protect blood lipid against oxidative damage.

Vegetables Results and conclusion of the study Type of study

Pearled Barley Pearled Barley shows inhibitory effects toward human LDL oxidation

In vitro

Potatoes Metanolic extract of potato is able to inhibit linoleic acid oxidation

In vitro

Tri-colored swiss chard The aorta lipid peroxidation was found to be decreased significantly

Animal study (Rat) - In vivo

Onion Onion is able to inhibit LDL oxidation and increase the resistance of LDL against oxidation in vitro

In vitro

Celery Decrease of liposomal peroxidation intensity in liver and blood of mice

Animal study (Mice) - In vivo

Parsley Reduction of the intensity of lipid peroxidation in mice liver and blood

Animal study (Mice) - In vivo

Vegetable Results and conclusion of the study Type of study

Bi-colored spinach

Spinach is able to influence lipid peroxidation in a beneficial manner

intervention study in healthy human

Blue savoy cabbage

Phenolic extracts from Blue savoy cabbage inhibit a lipid peroxidation in a linoleic acid

emulsion

In vitro

Tuscan Black cabbage

Tuscan Black cabbage shows a protective effect on the oxidation of lipoproteins

In vitro

Purpule Carrot

Drinking carrot juice decreases lipid peroxidation

Human study – In vivo

Soup ingredients Type of study

Quantity of soup intake

Duration of intervention

Results

Barley soup (Orzotto) Human study (single-arm)

250 g/d 2 weeks

Serum levels of oxidised LDL

decreased after consumption

2 kinds of soup with carrot, tomato, broccoli and olive oil

Human study - clinical control trial

300 mL/d 4 weeks

Gazpacho (80% crude vegetables : tomato, cucumber, pepper ; olive oil and other minor components)

Human study - clinical control trial

500 mL/d 14 days

Literatures which show that the similar vegetable soups can protect blood lipid against oxidative damage

Evaluation based on Criteria for scientific substantiationCriteria

1) The food or food component should be characterized

2) Substantiation of a claim should be based on human studies

2a) Study groups are representative of the target group

2b) Appropriate controls

2c) An adequate duration of exposure and follow up to demonstrate the intended effect

2d) Characterization of the study groups’ background diet and other relevant aspects of lifestyle

2e) An amount of the food consistent with its intended pattern of consumption

2f) The influence of the food matrix on the functional effect of food

2g) Monitoring of subjects’ compliance concerning intake of food

3) Markers are used when the true endpoint cannot be measured

4) Markers should be biologically and methodologically valid

5) A Change in target variable is statistically different and biologically meaningful

6) Taking into account the totality of the available data and by weighing of the evidence.

THANK YOU FOR ATTENTION

Synergistic effect

Phenolics Phenolic radical

Apart from phenolic compounds, the food product also contain vitamin C

- Phenolics give H atom to radical - Radicals Non-radicals- Phenolics Phenolic radicals- Ascorbic acid give H atom to Phenolic radicals- Ascorbic acid Dehydroascorbic acid- Phenolic radicals Phenolics

Phenolic compounds (<400 mg/day)

Food Quantity of phenolic compound

intake

Duration of Intervention

Biomarkers Subjects Type of study

soy germ pasta

31–33 mg/day isoflavones 8 weeks Decreased plasma

oxidized LDL

26 adults with T2D (13 men/13 women, aged 62 ± 7 years)

RCT

Pycnogenol (Extract)

150 mg/day proanthocyanidins 8 weeks Decreased F2-

isoprostanes

23 patients with coronary artery disease (CAD)

RCT

Cocoa drink 187 mg /day Flavanols < 1 day Decreased F2-

isoprostanes 20 Healthy subjects RCT

Grape-seedextract

300 mg/day total phenolic < 1 day - Decreased lipid

peroxide 8 Healthy subjects RCT

Phenolic compounds (400-500mg/day)

Food Intake Quantity Duration of Intervention

Biomarkers Subjects Type of study

Black tea

400 mg/day flavonoid(Catechins and theaflavins)

4 weeks

lag time before the initiation of LDL oxidation(Prolonged by 8 min (10-15%))

healthy male volunteers (n = 22) aged 22 ± 1 y(14 treatment, 18 control)

Clinical Control trial

cranberry juice

460 mg/day total phenolic 8 weeks

- Decreased plasma oxidized LDL - Decreased plasma malondialdehyde

Adults (n = 36) with features of metabolic syndrome

RCT

Green tea polyphenol

extract

480 mg/day catechin 2 weeks

lag time before the initiation of LDL oxidation(prolonged by 14 min)

22 male volunteers aged between 22 and 32 years

Clinical control trial

Catechin 500 mg/day catechin 4 weeks Decreased plasma

oxidized LDL

- 40 healthy adult volunteers (10 men, 30 women) - catechin group (n = 29)

Clinical control trial

Phenolic compounds (>500 mg/day)Food Intake Quantity Duration of

InterventionBiomarkers Subjects Type of

study

Pomegranate juice

570 mg/daytotal phenolics

until disease progression

Decreased lipid peroxide - Clinical

trial

Sweet potato leaves

902 mg/day polyphenols 2 weeks Decreased plasma

TBARS15 healthy male volunteers RCT

High purple sweet potato

leaves

902 mg/day polyphenols 2 weeks

- Prolonged lag time before the initiation of LDL oxidation

15 elite basketball players (6 men and 9 women, aged 20-24 y)

RCT

Perilla 1000 mg polyphenol/day 4 hr

- Prolonged lag time before the initiation of LDL oxidation - Decreased lipid peroxide - TBARS

Female healthy volunteers (n = 8)

RCT

Tart cherry juice

1100 mg polyphenol/day 2 weeks Decreased F2-

isoprostanes

12 healthy men and women (6 men, 6 women) aged 61–75 y

RCT

Supporting Evidence

Literatures which show that each vegetable ingredient can protect blood lipid against oxidative damage.

Vegetable

Method Results and conclusion of the

study

Type of study

Amount of vegetables used

in the experiment

Pearled Barley

Difference between the amount of formed diene in the control sample (antioxidant free) and in the presence of antioxidant barley extracts

Pearled Bareley shows inhibitory effects toward human LDL oxidation

In vitro Extracts prepared by weighing 1,0g wholegrain

Potatoes Antioxidant activities of potato extracts compared with a commercially available antioxidant (α-tocopherol)

Metanolic extract of potato is able to inhibit linoleic acid oxidation

In vitro total phenolic: 16,58 to 36,24 mg GAE/100g dry sample

Tri-colored swiss chard

Chard extract added in rat diet compared to a basic diet without chard

The aorta lipid peroxidation was found to be decreased significantly

Animal study (Rat) - In vivo

Chard extract (2g/kg) in 28 days

Vegetable Method Results and conclusion of the study

Type of study

Amount of vegetables used in the experiment

Onion Inhibitory effect of extracts of onion on LDL oxidation in vitro compared with shallot and garlic extracts

Onion is able to inhibit LDL oxidation and increase the resistance of LDL against oxidation in vitro

In vitro Onion concentration: 5 and 10 mg/mL

Celery Treatment with Celery leaves extracts compared to a control (no treatment)

Decrease of liposomal peroxidation intensity in liver and blood of mice

Animal study (Mice) - In vivo

1.0 mL/kg (i.p.) of celery leaves extracts at the same time.

Parsley Treatment with Parsley leaves extracts compared to a control (no treatment)

Reduction of the intensity of lipid peroxidation in mice liver and blood

Animal study (Mice) - In vivo

1.0 mL/kg (i.p.) of parsley

Purpule Carrot

Effects of drinking carrot juice on anthropometrics, blood Pressure, blood chemistry, lipid panel, hormones, inflammatory markers, and antioxidant status before and after the test

Drinking carrot juice decreases lipid peroxidation

Human study – In vivo

16 fl oz of daily freshly squeezed carrot juice for 3 months

Vegetable

Method Results and conclusion of the

study

Type of study

Amount of vegetables used

in the experiment

Bi-colored spinach

Comparaison between markers of lipid peroxidation

before and after a 10 days intake of spinach

Spinach is able to influence lipid

peroxidation in a beneficial manner

intervention study in healthy

individuals

6,2 g of spinach power contains

5mg/lutein

Blue savoy cabbage

Evaluation of the antioxidant capacity and content of Blue

savoye cabbage

Phenolic extracts from Blue savoye cabbage inhibit a

lipid peroxidation in a linoleic acid emulsion

In vitro2g and 0,5g/L of

linoleic acid emulsion

Tuscan Black

cabbage

Comparaison beteween lipid peroxidation levels of

oxidized lipoproteins without and with vegetable extracts at

different concentrations

Tuscan Black cabbage shows a protective

effect on the oxidation of lipoproteins

In vitro 5/10/25/50 ug/mL

Soup ingredients Type of study

Quantity of soup intake

Duration of intervention

Results

Barley soup (Orzotto) Human study (single-arm)

250 g/d 2 weeks Serum levels of oxidised LDL decreased after consumption

2 kinds of soup with carrot, tomato, broccoli and olive oil

Human study - clinical control trial

300 mL/d 4 weeks Serum levels of oxidised LDL decreased after consumption of both soups

Gazpacho (80% crude vegetables : tomato, cucumber, pepper ; olive oil and other minor components)

Human study - clinical control trial

500 mL/d 14 days Increase of serum vitamin C concentration

Literatures which show that the similar vegetable soups can protect blood lipid against oxidative damage